Synthetic-lethality screens, initially proposed by Hartwell, Friend and colleagues, hold great promise for cancer therapy since they enable the identification of drugs that selectively kill tumor cells while sparing normal tissue. This approac presumes that cancer cells harboring a specific mutation will be more sensitive to manipulation of certain pathways than normal cells that lack the mutation. The Von Hippel-Lindau (VHL) tumor-suppressor gene is mutated/lost in about 90% of Clear Cell Renal Cell Carcinomas (CC-RCCs). Because VHL-deficient CC-RCCs resist current therapies and frequently metastasize, identifying effective new therapies will be crucial for treatment of CC-RCC patients. By screening of the Library of Pharmacologically Active Compounds, I identified seven compounds that selectively kill VHL-deficient CC-RCC. Importantly the molecular targets that these compounds bind and inactivate are already known. This approach is innovative since it is based on a drug screen designed to identify not only compounds but also their respective protein targets that specifically affect VHL-deficient CC-RCC cells. In this application we will focus on two compounds (Y-27632 and RKI-1447) that display the strongest synthetic lethality with VHL deficiency. Since both of these compounds were reported to target Rho-associated protein kinases (ROCK1&2), we performed in vitro RNA-interference experiments and verified that down regulation of ROCK1, but not ROCK2, is critical for VHL-deficient CC-RCC death. We hypothesize that targeting the ROCK pathway in vivo will lead to suppression of VHL-deficient tumors with minimal toxicity to normal tissues. We further hypothesize that the activation of the Hypoxia-Inducible Factor (HIF) pathway downstream of VHL loss is important for the observed synthetic-lethal effect. We will test these hypotheses through the following specific aims: (1) To assess the in vivo efficacy of ROCK inhibition in an orthotopic mouse model of CC-RCC; (2) To determine if ROCK inhibition is synthetically lethal with Hypoxia-Inducible Factor (HIF) pathway in CC-RCC. Importantly, all VHL-deficient CC-RCC patients can be divided to two groups: those with tumors expressing both HIF1 and HIF2, and those with tumors expressing HIF2 only. If the observed synthetic lethality is mediated by HIF2, all VHL-deficient patients would be expected to respond to ROCK-targeted therapies; if the observed synthetic lethality is mediated by HIF1+HIF2, patients whose tumors lack HIF1 would be expected to be less responsive to ROCK-targeted therapies. It is therefore critical to determine whether the synthetic lethality depends on HIF, and if so, whether it depends on HIF1, HIF2, or both. Thus, the objective of the current study is to address the utility of ROCK inhibitors as candidate patient therapeutics and identify potential biomarkers that will predict whether patients will benefit from ROCK inhibition. Both of the above aims are expected to be clinically important and have a major impact on cancer therapies for CC-RCC.